Ras and related small molecular weight GTPases function in the regulation of signaling and cell growth, and collectively serve to control cell proliferation, differentiation and apoptosis [Tekai et al. 2001; Wennerberg et al. 2005]. The Ras-related GTPases are divided into four subfamilies with the Rab proteins regulating membrane transport, Rho proteins (including Rac and Cdc 42) regulating cytoskeletal rearrangements and responses to signaling, Arf/Sar proteins regulating membrane and microtubule dynamics as well as protein transport, and Ran proteins controlling nucleocytoplasmic transport. This project focuses on representative Ras, Rho, and Rab family members to validate the approach for the identification of new chemical compounds with novel therapeutic potential in cell signaling and growth control.

Ras and Ras-related GTPase functions are tightly regulated, and dysregulation is causal in a wide variety of human diseases. Ras mutations resulting in impaired GTP hydrolysis and plasma membrane hyperactivation are linked to many human cancers [Farnsworth et al. 1991; Sukumar et al. 1983; Taparowsky et al. 1982; Boylan et al. 1990; Hruban et al. 2004; Abrams et al. 1996]. Point mutations in the Rab and Rho GTPases are also causal in diverse human diseases affecting pigmentation, immune, and neurologic functions [Houlden et al. 2004; Verhoeven et al 2003; Williams et al. 2000; Bahaderan et al. 2003; and preliminary findings]. Rab and Rho mutants identified in human disease act as dominant negatives either due to a failure to bind GTP or due to inappropriate coupling of the active proteins with downstream effectors. To date, inhibition of Ras and Ras-related proteins has relied largely on altering membrane recruitment with various drugs affecting prenylation [Morgillo F and Lee HY, 2006; Russell RG, 2006; Park, et al. 2002]. Generally, Ras proteins must be farnesylated for proper membrane localization, while Rab and Rho proteins are geranylated. Such strategies lack specificity and are problematic because each of these prenylation machineries is required for the proper function of many Ras superfamily members. Rational drug design has only recently been applied to identify the first two small molecule inhibitors of Rho GTPase family members [Gao, et al. 2004; Nassar et al. 2006]. Therefore, broadly testing the Ras-related GTPases as targets for small molecule inhibitors and activators is expected to identify new classes of compounds that may be useful in the treatment of human disease, as well as in unraveling the molecular details of how Ras-related GTPases function.

The primary HTS assay was a no-wash fluorescent GTP-binding assay adapted to multiplexed, high-throughput measurements whereby multiple GTPases were simultaneously screened against the MLSCN library. The specificity is based on the observation that individual GTPases including wt and activated forms exhibit measurably distinct affinities for Bodipy-FI-GTP vs GTP. The assay involves the binding of fluorescent GTP to G protein-GST fusion proteins on GSH beads. A set of six G proteins (Rac 1 wt, Rab7 wt, Rac 1 activated, Ras wt, Rab 2 wt, Cdc42 wt) are arrayed under conditions of divalent molecule depletion. The primary screen was performed in 384-well format with 194,656 compounds at a compound concentration of 10 microM to identify 398 potential small molecule inhibitors of each of these GTPases

Compounds that satisfied hit selection criteria in the primary screen (change in % activity greater than 20% from baseline) were evaluated via dose response to confirm activity and determine potency. The active compounds from these dose responses were then used to develop structure activity relationships for the different GTPases.

Based on the original inhibition hit MLS000091369, a synthetic plan was carried out by the University of Kansas Specialized Chemistry Center exploring both substitution on the peripheral phenyl groups as well as modification to the chain length between the oxadiazole and the piperazinyl-carboxamide linker. Later modification to the substructure core included exploring other heterocycles in place of the oxadiazole as well as replacement of the piperazine with piperidine.

Bead sets are coated with individual GST-small G proteins, blocked with Buffer (0.01% NP-40; 30mM HEPES pH 7.5; 100mM KCl; 20mM NaCl; 1mM EDTA; 0.1% BSA and 1mM DTT), incubated overnight at 4 degrees C, and finally washed in buffer. The different bead sets, acquired from Duke Scientific, have similar size (~ 4 microm diameter) however they are distinguished by varied magnitude of emission at 665 +/-10 nm with excitation at 635 nm.Test compounds were serially diluted 1:3 eight times for a total of nine different test compound concentrations in DMSO. Final compound dilutions in DMSO ranged from 1 microM to 10mM. These dilutions were then diluted 1 to 100 to give an assay concentration range of 10 nanoM to 100 microM.The assay is conducted in 384-well microplates in a total well volume of 10.1 microliters (5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 200nanoM Bodipy-FL-GTP in buffer containing BSA and DTT for a final concentration of GTP of 100nanoM). Positive Controls, which contain bead mixture and fluorescent GTP but no test compound, are located in columns 1 and 2 on plate. Negative Controls containing bead mixture with fluorescent GTP and 0.5 mM unlabeled GTP, are collected from a separate test tube. Plates are placed on rotators and incubated for 40-45 minutes at 4 degrees C. Dose response experiments reported here include one multiplex format for: Rab7 wt, Rab2 wt, H-Ras wt, H-Ras constitutively active, Cdc42 wt, and Cdc42 constitutively active, and 2 single-plexes for: Rac1 wt and Rac1 constitutively active. Sample acquisition and preliminary analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt system interfaces a flow cytometer and autosampler for high-throughput microliter volume sampling from 384-well microtiter plates [Kuckuck, et al., 2001; Ramirez, et al., 2003]. The stream of particles is excited at 488 nm and 635 nm, and flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and emission at 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Analysis of the time-resolved acquisition data file uses IDLeQuery/HyperView software to merge the flow cytometry data files with compound worklist files generated by HyperSip software. Gating based on forward scatter (FS) and side scatter (SS) parameters is used to identify singlet bead populations. Gating based on FL8 emission distinguishes the beads coated with different proteins, and the green median fluorescence intensity (MFI) per bead population (well) is calculated. The raw data are parsed in IDLeQuery/HyperView to produce annotated fluorescence summary data for each well. The parsed data are then processed through an Excel template file constructed specifically for the assay to fit the data via GraphPad Prism. Calculations:In dose response experiments, the percent response was calculated for each GTPase target by the following equation:%Response = (SampleMFI - NCntrl)/(PCntrl - NCntrl)where SampleMFI is the median fluorescence intensity of the compound sample, NCntrl is the median fluorescence intensity measured in the presence of access blocking non-fluorescent GTP, and PCntrl is the median fluorescence intensity measured in the presence of DMSO. The %Response was 100% for compounds not affecting the binding of Bodipy-FL-GTP, and greater than 100% if the compound increased the binding of Bodipy-FL-GTP and less than 100% if the compound decreased the binding of Bodipy-FL-GTP.The % response values for the entire concentration range of a test compound were fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the following parameters of the model: EC50, microM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.Assessment of the validity of the estimates from the Prism fits was filtered via the following criteria:- -8 < LOGEC50 < -4 (the computed EC50 value should be in the interval of tested concentrations)- STD_LOGEC50/|LOGEC50| <0.15 (the standard error of LOGEC50 should be no greater than 15% of the absolute value of LOGEC50)- 0.5 < |HILLSLOPE| < 2 (the absolute value of HILLSLOPE should be higher than 0.5 and lower than 2)For compounds with significant inhibitory affects (i.e., Hillslope < 0), Ki values were calculated from the EC50 values based on the following equation:Ki = EC50/(1 + [Bodipy-FL-GTP]/Kdwhere [Bodipy-FL-GTP] is the assay concentration of fluorescently labeled GTP of 100 nanoM and Kd is the specific targets affinity for GTP. In-house determination of the different Kds are as follows: Rac wt 10.3 nanoM, Rab7 wt 7.0 nanoM, Cdc42 act 18.3 nanoM, Cdc42 wt 26.8 nanoM, Ras wt 18.5 nanoM, Rab2 wt 6.8 nanoM, Ras act 8.0 nanoM. Note that this equation is theoretically only applicable to dose response curves of absolute value of Hillslopes equal to 1, however the calculations of Ki's reported did not take into account the Hillslope.PUBCHEM_ACTIVITY_SCORE were calculated based on an EC50 cutoff of 30 microM, by using the following equations:SCORE = 100*(1-EC50/30 )And compounds were demeaned active if the EC50 is equal to or less than 30 microM and the span of the %Response is greater than or equal to 25%. Span is the magnitude of %Response change over the tested concentration range based on either fitted values of TOP minus BOTTOM or from %Response values at lowest to highest concentration. An active compound have a PUBCHEM_ACTIVITY_SCORE greater than 0.Keywords: NIH Roadmap, NMMLSC, high throughput flow cytometry,Rac1 wt, Rab7 wt, Rac1 activated, Ras wt, Ras activated, Rab2 wt, Cdc42 wt, Cdc42 activated, multiplex, bead-based, screening, dose response, SAR